Tick-Borne Encephalitis Virus, United Kingdom

Maya Holding; Stuart D. Dowall; Jolyon M. Medlock; Daniel P. Carter; Steven T. Pullan; James Lewis; Richard Vipond; Mara S. Rocchi; Matthew Baylis; Roger Hewson

Disclosures

Emerging Infectious Diseases. 2020;26(1):90-96. 

In This Article

Methods

Sample Collection

We recruited persons involved in routine management of deer from across the United Kingdom to collect serum and tick samples from any species of deer. This program was promoted through organizations involved in deer management. These deerstalkers submitted 1,323 serum samples (and tick samples where present) from deer culled in England and Scotland during February 2018–January 2019. The University of Liverpool Ethics Committee (ref: VREC596) granted ethics approval for this study on February 1, 2018.

Blood samples were collected in serum-separation vacutainers from the chest cavity during gralloching, and blood-fed ticks were collected from any location on the deer carcass. Samples were centrifuged at 1,500 relative centrifugal force for 10 min and aliquoted. Serum and tick samples were stored at −80°C until further processing.

ELISA Testing

We tested serum samples for antibodies to TBEV using the commercial Immunozym FSME IgG All Species ELISA (Progen, https://www.progen.com) according to the manufacturer's instructions. We read plates at an optical density ratio of 450 nm. We considered samples with a reading of ≥127 Vienna units/mL to be seropositive.

Hemagglutination Inhibition Testing

We tested serum samples for antibodies to LIV using a hemagglutination inhibition (HAI) test.[17,18] We considered samples with a titer ≥20 seropositive. A small number of samples did not have sufficient serum for HAI testing.

Tick Identification and RNA Extraction

We morphologically identified all ticks collected from culled deer within a 15-km radius of any TBEV ELISA–seropositive deer[19] to life stage and species level. We individually homogenized the ticks in 300 μL RLT buffer (QIAGEN, https://www.qiagen.com) in MK28-R Precellys homogenizing tubes using a Precellys 24 homogenizer (Bertin, https://www.bertin-instruments.com) at 5,500 rpm for 5 sec, followed by a 30-sec break; we repeated this process 4 times. We then added 300 μL of isopropanol and passed the tick homogenate through a QIAshredder (QIAGEN). We extracted total RNA using the BioSprint 96 One-For-All Vet Kit (QIAGEN) and eluted it into 100 μL AVE buffer according to the manufacturer's instructions.

rRT-PCR

We tested individual tick samples for LIV/TBEV RNA using a sensitive LIV/TBEV assay.[20] We amplified RNA in 20 μL rRT-PCR mix containing 0.8 μL Invitrogen (https://www.thermofisher.com) Superscript III/Platinum Taq Mix, 10 μL Invitrogen 2X reaction mix, 1.6 μL 50 mmol/L MgSO4,, 1 μL of 1 μmol/L forward primer, 1 μL of 18 μmol/L reverse primer, 0.2 μL of 25 μmol/L probe, 5 μL template, and 0.4 μL molecular-grade water.

We also tested all RNA-positive samples using a secondary assay designed to detect only LIV.[21] We amplified RNA in 20 μL rRT-PCR mix containing 0.8 μL Invitrogen Superscript III/Platinum Taq Mix, 10 μL Invitrogen 2X reaction mix, 0.8 μL of 10 μmol/L forward primer, 1.8 μL of 10 μmol/L reverse primer, 1.0 μL of 5 μmol/L probe, 5 μL template, and 0.6 μL molecular-grade water.

Sequencing and Phylogenetic Analysis

We prepared the tick sample that showed a high level of TBEV RNA for metagenomic RNA sequencing[22] and assembled the sequencing data using SPAdes version 3.1.1.[23] We inferred the evolutionary history by using the maximum-likelihood method based on the Tamura 3-parameter model.[24] We used the tree with the highest log likelihood. We automatically obtained initial trees for the heuristic search by applying neighbor-joining and BioNJ[25] algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. The analysis involved 10 full-length genomic TBEV nucleotide sequences and was performed using Molecular Evolutionary Genetics Analysis version 7.0 software.[26]

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